A basal lamina is constituted by an extracellular matrix component which is mainly a collagen, and exists universally in an organism. One of macromolecular proteins constituting the basal lamina is laminin (hereinafter, often abbreviated as LN). The LN is classified into more than ten kinds based on the structure thereof, which are different in functions and localized tissues, and is classified by adding a number at the end, e.g., LN1 or LN2. Every LN is constituted by a complex formed from three polypeptide chains each having a different amino acid sequence, and gives a cross-like molecular form in an electron microscope observation. The three polypeptide chains are respectively called α chain, β chain, and γ chain, and each has several molecular species (α1 to α5, β1 to β3, and γ1 and γ2).
In the basal lamia between epithelial cells and a connective tissue backing the epithelial cells, there exists a cell adhesion structure peculiar to the epithelial cells. As an extracellular matrix-constituting protein which exists mainly in the above structure, laminin 5 is known (hereinafter, often abbreviated as LN5). Physiological characteristics of LN5 is that it is the only LN in the above LN group, produced solely from epithelial cells and that it has an activity to promote adhesion to the basal lamia and motility of epithelial cells. As for the epithelial cell, it is known phenomena that it adheres strongly to LN5 and the basal lamia containing LN5 via a specific receptor called integrin on a cell membrane of the epithelial cell itself, and that it migrates aggressively.
The LN5 is constituted by a complex formed from one α3 chain, one β3 chain, and one γ2 chain. The γ2 chain, in particular, is considered to be specific to LN5, and is not included in the other LN molecular species [Dev. Dyn., 218, 213-234 (2000)]. In addition, it is found that LN5 is partially degraded by a proteolytic enzyme (protease) when secreted from the epithelial cells, and it is indicated that, especially by shedding an N-terminal part of the γ2 chain, the remaining molecular entity of LN5 from which the N-terminal part was released has an increased activity for promoting cell movement [J. Cell Biol., 148, 615-624 (2000)].
That is, the amount of LN5 γ2 chain fragments released from the epithelial cells, etc. reflects an LN5 production amount in the cells, and it considered to be a potential index for measuring an epithelial cell motility-promoting activity of LN5 in an epithelial tissue.
Recently, a lot of study results reported that an expression of the LN5 increased in a malignant tumor tissue from epithelial cells. There are many reports, in particular, that the LN5 expression level correlates well with an invasiveness of a malignant tumor, and its application as a pathological marker for the purpose of in vitro diagnosis of cancer is considered to be possible [J. Natl. Cancer Inst., 91, 1882-1887 (1999) and Cancer, 85, 2315-2321 (1999)].
However, in the above studies, there is mainly employed a method in that a pathologic tissue is removed from a body of a patient through an operation to prepare a section, and then an expression site of a target protein is immunstained with an antibody. Such method lacks quantitativeness and versatility in terms of practical application to in vitro diagnosis in medical places, and also has a problem of imposing physical strains on a patient, for example, a surgical operation and a tissue biopsy. Therefore, an establishment of a simple and quick method of determining a level of an LN5 antigen in a biological sample, such as blood, which can be sampled relatively safely is desired.
Recently, there has been reported an enzyme-linked immunosorbent assay (ELISA) using two kinds of monoclonal antibodies to LN5 (a monoclonal antibody BM165 against an α3 chain and a monoclonal antibody 6F12 against a β3 chain) [J. Immunol. Meth., 224, 161-169 (1999)]. However, this report shows no example that an LN5 antigen in a biological sample is detected by ELISA.